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. 2020 Apr 18;52(2):214–220. [Article in Chinese] doi: 10.19723/j.issn.1671-167X.2020.02.004

角蛋白18与剪切因子PSF相互作用介导PSF的膜易位并维持髓系白血病细胞的化疗敏感性

Interaction between PSF and cytokeratin 18 mediates PSF relocation to cell membrane and maintains chemosensitivity of myeloid leukemia

Si-mei REN 1,2,△, Lu-yao LONG 1,2, Cheng-shan XU 1
PMCID: PMC7433459  PMID: 32306001

Abstract

Objective

To identify the chaperone of polypyrimidine tractor-binding protein-associated splicing factor (PSF) in myeloid leukemia cells, and to explore the mechanism and redistributive pattern to cell surface of PSF in chemo-sensitive HL60 cells and resistant HL60/DOX cells.

Methods

The eukaryotic expression vector of PSF was transfected with liposomes transiently, then flow cytometry was used to detect the expression level of PSF on the cell surface 24 h, 48 h and 72 h after vector transfections. We constructed a chimeric expression vector, streptavidin binding peptide (SBP)-PSF, meanwhile this vector was transfected and made SBP-PSF fusion protein overexpress. In addition, we used streptavidin magnetic beads to precipitate the cellular chaperonin of PSF and then identified its chaperonin by mass spectrometry (MS). Lentiviral vectors containing cytokeratin18 (K18) interference sequences were transfected into 293T cells to prepare lentivirus. HL60 and HL60/DOX cells were infected with lentivirus to obtain stable interfering K18 cell lines. Next, flow cytometry was used to test the membrane relocation level of PSF. Together, these methods confirmed the similar or different mechanisms of the PSF redistributing to membrane synergistically mediated by K18 in HL60 and HL60/DOX cells.

Results

The expression of membrane relocated PSF was detected every day for three days(at the end of 24 h,48 h and 72 h) after transient overexpression. The expressing rate of PSF on the cell surface was 22.4%±3.5%, 37.9%±6.0%, 58.3%±8.8%, respectively in sensitive HL60 cells, while that was 4.7%±0.5%, 3.9%±0.6%, 2.9%±0.6% , respectively in resistant HL60/DOX cells. The difference of expressing rate on each day was significant, P<0.01. We identified K18 detected by co-immunoprecipitation and mass spectrum assay which was the cellular chaperone of PSF. We found that K18 knockdown decreased the PSF expression level which redistributed on cell surface from 48.9%±5.4% to 6.2%±1.0% in sensitive HL60 cells, and from 9.11%±1.2% to 2.21%±0.51% in resistant HL60/DOX cells, respectively.

Conclusion

K18 is the intracellular chaperonin of PSF. The interaction of PSF and K18 mediates its redistribution to cell membrane in sensitive cells. While in resistant cells, PSF failed to relocate at the cell surface and accumulated in cells, which mediated resistance to chemotherapeutics.

Keywords: Tumor multidrug resistance, Polypyrimidine tract-binding protein-associated splicing factor, Cytokeratin 18

肿瘤的耐药性是导致患者预后差的根源,耐药的发生是多种分子历经多个阶段的复杂生物学过程,这些分子在表达,定位,修饰,下游调控等多种细胞事件中发生改变,彼此依赖又相互制约.近年来对新的耐药相关调控蛋白的研究发现,肿瘤细胞核和细胞浆内某些蛋白的易位表达与肿瘤耐药的发生密切相关[1].我们前期通过一株特异性单抗鉴定到一个全新的耐药相关蛋白----多嘧啶结合蛋白相关的剪切因子(polypyrimidine tract-binding protein-associated splicing factor, PSF)[2].有趣的是,在前期研究中我们发现该蛋白作为一个经典的细胞核蛋白可以异常定位到细胞膜上表达,并且其在敏感和耐药的急性早幼粒细胞膜表面易位表达的水平与化疗敏感性相关.

PSF是一个多脯氨酸和谷氨酰胺酶的前mRNA剪切因子,全长707个氨基酸,定位于1号染色体.它在细胞内介导广泛的蛋白质-蛋白质和蛋白质-核酸相互作用,总体上起到了多功能分子支架的作用;其功能涉及细胞转录调节,DNA损伤修复,昼夜节律,细胞凋亡和周期调控等[3].RNA剪切是基因表达调控中的重要过程,极大拓展了基因组转录的多样性.近年来剪切因子异常状态在各种疾病发生,进展和治疗反应性中的作用受到越来越多的关注,在血液病和实体瘤中的报道尤其多[4,5,6].PSF在肾癌,乳腺癌,前列腺癌,淋巴瘤中的各种作用已相继被报道.在既往研究中,PSF常作为免疫共沉淀的伴侣蛋白被纯化出来,大多时候被当做杂质或被作为复杂蛋白复合物和胞内诸多反应中的被动参与者[7].但近年的研究表明,PSF在维持基因组完整性和平衡细胞内各种活性,维持细胞稳态中发挥独特且关键的作用,是胞内诸多重要功能的整合者,甚至是疾病进程的始动者[7].PSF在肿瘤发生发展中介导的各种复杂作用与其胞内伴侣蛋白密切相关,因而阐明PSF和其伴侣蛋白在各种功能中的调节作用对疾病诊疗有重要价值.

PSF结构域复杂,已被报道的在胞内可发生相互作用的伴侣蛋白包括p54nrb/NONO,RAD51,RAB23,SMN2,Annexin A2,STAT6,EWS等[8].我们之前报道的PSF易位细胞膜表达与维持化疗敏感性的关系是否由其已发现的伴侣蛋白所介导尚不可知[2].PSF在敏感株中易位到细胞膜的具体机制,其是否被动积累在耐药株的胞内以介导细胞耐药性的具体机制均不清楚.探究PSF易位细胞膜的机制对深入了解这一类分子所介导的耐药作用,以及其在肿瘤发生和进展中的作用,为进一步将之开发为诊断和治疗肿瘤耐药性的临床可用靶点均具有重要的科学意义.本文将通过设计的多种表达载体鉴定PSF的伴侣蛋白,并阐明敏感和耐药细胞株中PSF由胞内向细胞膜迁移的特征和机制.

1. 材料与方法

1.1. 试剂与载体

RPMI1640培养基,胎牛血清购自美国Gibco公司,鼠抗人PSF,抗人角蛋白18 (cytokeratin 18,K18)抗体购美国Sigma公司;鼠抗人GAPDH抗体购自ProteinTech公司,羊抗鼠IgG-HRP购自北京中杉金桥生物技术有限公司;RNA提取试剂Trizol,Dynabeads M-280 (11205D) 链亲和素磁珠,脂质体Lipofectamine 3000,Alexa633标记的抗IgG二抗购自美国ThermoFisher公司;逆转录试剂盒,实时荧光定量PCR试剂购自美国Promega公司.pLVTH-shK18干扰载体和pLVTH-shControl对照载体由香港科技大学神经分子国家重点实验室黄平波教授馈赠[9], psPAX2和pMD2G-VSVG载体购自北京诺为生物科技有限公司.

1.2. 细胞系与细胞培养

HL60细胞系和多柔比星耐药细胞株HL60/DOX细胞系由日本北海道大学医学院Masahiro Imamura教授馈赠[2],并于国家卫健委临床检验中心血液室保存.用含10%(体积分数)灭活胎牛血清的RPMI1640 培养基于37℃,5%(体积分数) CO2 孵箱中常规培养.取对数生长期的细胞用于实验,耐药细胞停药一周后用于实验.

1.3. 细胞RNA提取和逆转录实时荧光定量PCR (reverse transcription and real-time quantitative polymerase chain reaction,RT-qPCR)

收集细胞,移至无RNA酶的EP管中,按照每107个细胞加入1 mL Trizol试剂的比例裂解细胞.参照说明书方法提取细胞总RNA.按照逆转录试剂盒说明书配制反应体系,每20 μL体系中加入2 μg的RNA作为模板,每份样本设3个复孔.取3 μL逆转录产物用于qPCR实验, Bio-rad iQ5用于扩增反应.扩增条件为95℃ 10 min, 95° 15 s, 60 ℃ 40 s, 72 ℃ 30 s, 40个循环.计算每个样本的平均Ct值,以GAPDH作为内参基因,计算目的基因和内参基因的相对表达量,采用 2-ΔΔct法计算待比较的两组间的相对表达倍数.引物序列如下:GAPDH上游引物:5'-TACATGGTTTACATGTTC-3',下游引物:5'-GCCAGTGGACTCCACGAC-3';PSF上游引物:5'-GGATCCACGGGAAAGAGACA-3',下游引物:5'-CACTCATGGTTGCTGGTGG-3';K18上游引物:5'-TCTTGGTGATGCCTTGGAC-3',下游引物:5'-CCTGCTTCTGCTGGCTTAAT-3'.

1.4. 细胞总蛋白提取与Western blot法检测PSF表达

PBS洗涤细胞两次,收集细胞沉淀,按1 mL/107细胞加入蛋白裂解液RIPA,冰浴缓摇20 min;13 000 r/min, 4 ℃连续离心15 min,收集上清加上样缓冲液.十二烷基硫酸钠聚丙烯酰胺凝胶(SDS-polyacrylamide gel electrophoresis, SDS-PAGE)电泳后将凝胶上的蛋白转移至聚偏二氟乙烯(polyvinylidene fluoride,FVDF)膜上, 5%(质量分数)脱脂奶粉室温封闭2 h,加一抗4 ℃ 孵育过夜.次日用1×TBST缓冲液洗膜一次,加入二抗,室温孵育1.5 h,1×TBST缓冲液洗膜3次,蛋白显影,GAPDH作为内参.

1.5. 免疫共沉淀发现伴侣蛋白

构建含有链亲和素结合肽(streptavidin binding peptide,SBP)和PSF融合蛋白的阳性载体pcDNA3.0-SBP-PSF,对照组载体只插入含有SBP多肽的片段,命名为pcDNA3.0-SBP.分别向HL60细胞转染阳性载体和对照载体,常规培养3 d后提取细胞总蛋白,以备做免疫共沉淀(co-immunoprecipitation,CO-IP).向Dynabeads M-280中加入预冷的RIPA裂解液,4 ℃缓摇30 min,13 000 r/min,4 ℃离心3 min,预处理磁珠.转染pcDNA3.0-SBP-PSF组和转染pcDNA3.0-SBP组的细胞总蛋白裂解液中分别加入10 μL预处理过的Dynabeads M-280,旋转混合仪中4 ℃反应3 h.13 000 r/min瞬时离心10 s,弃上清,收集沉淀复合物.预冷的PBS缓冲液洗涤沉淀复合物3遍,每次10 min.之后加2×上样缓冲液重悬免疫沉淀复合物,煮沸5 min,吸取上清,进行SDS-PAGE分析.

1.6. 质谱鉴定

将SDS-PAGE中的目的条带仔细切下,送北京华大蛋白质研发中心有限公司进行基质辅助激光解析电离飞行时间质谱(matrix-assisted laser time of flight mass spectrometry, MALDI-TOF-MS)分析,结果与Swissprot No.P23246蛋白数据库比对.

1.7. 基因载体转染与慢病毒感染

利用真核表达载体pCDNA3.0构建含有HA标签蛋白,甘氨酸-丝氨酸柔性短肽和PSF全长的融合基因片段"HA-G4S-PSF"的载体pCDNA3.0-HA-G4S-PSF,按脂质体LipofectAMINE 3000说明书推荐的方法转染细胞,继续加入含10%胎牛血清的RPMI1640培养过夜后,于0,24,48,72 h收集细胞检测膜HA表达水平.培养293T细胞至适宜密度,使用LipofectAMINE 3000按照3 ∶1 ∶4的比例向293T细胞中同时转染psPAX2,pMD2G-VSVG和pLVTH-shK18/pLVTH-shControl三种载体.次日更换新的培养基继续培养3 d,收集干扰K18的慢病毒液的上清浓缩分装,-80℃冻存.细胞按5×104个/mL接种2 mL至6孔板,加入病毒液20 μL[感染指数(multiplicity of infection,MOI)=10],同时加入聚凝胺试剂Polybrene至终浓度5 mg/L.感染过夜后更换新的培养基,72~96 h后用终浓度5 mg/L的嘌呤霉素连续加药两天,筛选感染阳性克隆,继续培养,停药4 d后的细胞用于后续实验.

1.8. 免疫荧光染色和细胞膜蛋白PSF的表达分析

收集对数生长期的细胞,用PBS洗涤两次,加入适量的HA抗体或PSF抗体和IgG1a同型对照抗体,4 ℃避光孵育40 min,PBS洗一次.加入兔抗鼠IgG-FITC或IgG-APC二抗,4 ℃避光孵育20 min,PBS洗两次.细胞重悬于PBS缓冲液,绸布过滤,流式细胞仪检测细胞膜HA或PSF表达水平.

1.9. 统计学分析

各组实验独立重复3~5次,采用SPSS 21.0进行统计学分析,数据以均数±标准差表示,两组间比较采用two-tailed Student's t检验,P<0.05为差异具有统计学意义.

2. 结果

2.1. 敏感和耐药HL60细胞的一般特性及PSF在两株细胞中的表达水平

敏感的HL60和耐药的HL60/DOX细胞系的生长曲线如图1A所示,在未给予任何干预情况下,敏感株增殖能力强于耐药株,差异有统计学意义(P<0.001).HL60/DOX的耐药倍数为敏感株HL60的50.44倍.RT-qPCR验证PSF表达,采用2-ΔΔct法计算两株细胞间PSF mRNA的相对表达倍数.耐药株PSF表达水平为敏感株的(1.46±0.34)倍,两组间差异无统计学意义(n=5, t=1.38,P=0.20);总蛋白水平在两株细胞中基本一致,耐药株略高(图1B).

1.

HL60和HL60/DOX细胞的特征

Characteristics of HL60 and HL60/DOX

A, cell viability of HL60 and HL60/DOX cells were detected by MTS assay; B, transcriptional and protein expression level of PSF in HL60 and HL60/DOX cells.

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2.2. 流式细胞术检测PSF向细胞膜迁移的能力

瞬时转染含有鉴定标记HA标签蛋白,PSF和分选标记GFP的融合蛋白载体(图2A),在转染0,24,

2.

PSF向细胞膜迁移的能力

Ability of PSF to migrate to the cell membrane

A, fragment inserted at a polyclonal site of pcDNA3.0 vector and working panel of experiment; B, HL60 and HL60/DOX were transfected with pcDNA3 vector containing HA-PSF fusion fragment, PSF expression on cell surface was detected by flow cytometry (△P<0.01). PSF, polypyrimidine tract-binding protein-associated splicing factor; PSF-FL, PSF full length.

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48,72 h连续检测细胞膜HA表达水平.HA的膜表达水平代表了PSF由胞内向细胞膜迁移的量,4个时间点的表达水平如下:HL60分别为3.7%±0.7%,22.4%±3.5%,37.9%±6.0%,58.3%±8.8%;HL60/DOX别为1.9%±0.5%,4.7%±0.5%,3.9%±0.6%,2.9%±0.6%, 各时间点下HL60组和HL60/DOX组比较,差异均有统计学意义 (图2B, P<0.01).

2.3. 免疫共沉淀与质谱鉴定PSF伴侣蛋白

Dynabeads M-280磁珠经预处理后,分别与转染pcDNA3.0-SBP-PSF阳性载体和pcDNA3.0-SBP对照载体的HL60细胞总蛋白裂解液孵育,沉淀复合物变性裂解后经SDS-PAGE电泳分离.与对照载体组比较,阳性载体组可见相对分子质量40 000~66 000处有一蛋白条带(图3A);在耐药株HL60/DOX中行相同的实验,未见该位置处的蛋白条带.图3A中箭头所示条带切胶,送华大蛋白质研发中心进行MALDI-TOF-MS分析,与NCBI20110323数据库比对,结果显示13条经酶解的肽段与K18匹配,序列覆盖率为26%(图3B).K18肽指纹图谱鉴定,该蛋白相对分子质量47 000,NCBI检索序列号为gi|30311.进一步通过免疫共沉淀与Western blot行互惠验证,发现由SBP-PSF融合蛋白钓取的蛋白复合物可由K18单抗杂交显影;由K18单抗钓取的蛋白复合物也可由PSF单抗杂交显影(图3C), 此证明PSF和K18在细胞内确实存在相互作用.

3.

K18是PSF在胞内的伴侣蛋白

K18 is the intracellular chaperone of PSF

A, HL60 cells were transfected with SBP-PSF-pcDNA3.0 vector. Avidin magnetic beads were used for immunoprecipitation from whole lysates of HL60 cells. The precipitated proteins (arrow) were separated by 12% SDS-PAGE and visualized by Commassie blue staining; B, protein precipitation complexes were identified by mass spectrometry; C, reciprocal experiment between CO-IP and Western blot. CO-IP, co-immunoprecipitation; PSF, polypyrimidine tract-binding protein-associated splicing factor; SBP, streptavidin binding peptide; K18, cytokeratin 18.

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2.4. 下调K18对PSF膜迁移能力的影响

通过RT-qPCR鉴定了敏感株HL60和耐药株HL60/DOX中K18的转录水平,图4A显示耐药株较敏感株表达水平高,差异有统计学意义 (t=2.52, P<0.05).用干扰K18的慢病毒液分别感染HL60和HL60/DOX细胞,感染4 d后,经RT-qPCR鉴定,K18被有效敲减 (图4B,t=9.1, P<0.001).用GFP作为shK18病毒感染阴性和阳性的设门标记,用抗APC标记目的蛋白PSF,分析两株细胞中敲减K18与否对细胞膜上PSF表达水平的影响.图4C见敏感的HL60细胞株中,高表达GFP的克隆(P3门)即成功敲减K18的克隆,其细胞膜上PSF的表达水平显著降低(P5门峰左移);而耐药株HL60/DOX中,敲减K18与否对细胞膜PSF表达水平几乎无影响.PSF细胞总体易位水平由P4或P5门的阳性百分率除以P1门的阳性百分率所得.敏感株敲减前后的膜易位水平为48.9%±5.4% vs. 6.2%±1.0%;耐药株敲减前后的膜易位水平为9.11%±1.2% vs. 2.21%±0.51%.由此说明K18在敏感株中介导PSF由胞内向细胞膜的迁移,而在耐药株中,此功能障碍或缺陷.实验结果由3次重复实验所得,流式细胞结果展示其中之一.

4.

K18干扰后PSF在细胞膜上的表达分析

PSF membrane expression assay after K18 interference

A, transcriptional expression levels of K18 in sensitive and resistant HL60 cells, *P<0.05; B, K18 transcriptional expression levels after K18 knockdown in HL60 cells,# P<0.001; C, PSF expression on cell surface after K18 knockdown were detected by flow cytometry. PSF, polypyrimidine tract-binding protein-associated splicing factor; K18, cytokeratin 18.

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3. 讨论

近年来PSF在肿瘤细胞中的报道逐渐增多,PSF作为许多基因的调节剂在不同瘤种,不同的肿瘤微环境下既可以是肿瘤的抑制因子,也可以是原癌基因.肿瘤细胞的增殖和迁移,肿瘤患者的不良预后和PSF表达丰度的改变密切相关[10,11].PSF表达水平升高促进结直肠癌的生长增强侵袭性,并可通过雄激素受体介导的活性促进前列腺癌进展[6].在乳头状肾癌,神经母细胞瘤和白血病中可检测到PSF和TFE3,ALB1的融合表达[12,13].文献报道肿瘤细胞所处的微环境及肿瘤细胞对化疗的反应性也与PSF的调节密不可分[14], 可见PSF对肿瘤细胞的调节能力是多方面的,从细胞内事件到肿瘤细胞所处的微环境都有参与,这与PSF剪切RNA的能力和其在细胞内复杂而广泛的定位不无关系.我们前期研究[2]发现PSF可在细胞膜表面异常定位,并且敏感的血液肿瘤细胞膜表面的表达水平显著高于耐药株,提示这种易位细胞膜的表达模式可能和维持化疗敏感性相关.关于PSF异常定位到细胞膜表达,也有研究者曾在人的骨骼肌细胞和血管内皮细胞的细胞膜上发现PSF的表达,但均未探究其易位表达的机制和意义[15].

本文中,我们选取了急性早幼粒白血病细胞系HL60的敏感和耐药株.图1结果表明,敏感株PSF的RNA和蛋白水平都略低于耐药株,而我们之前的研究[2]已证明在这对细胞系中,PSF在敏感株细胞膜上的表达水平显著高于耐药株,这提示PSF在敏感株中由胞内向细胞膜的迁移能力显著强于耐药株.PSF在敏感株中是否主动向细胞膜迁移?在耐药株中是否由于某些蛋白功能障碍导致PSF在胞内发生了被动积累从而介导了细胞耐药呢?基于这种假设,我们构建了HA和PSF的融合表达载体,瞬时过表达HA-PSF融合蛋白后连续检测细胞膜上HA的表达水平,HA水平反映了过表达的PSF由胞内向细胞膜迁移的能力.我们发现敏感株中过表达的PSF随时间积累不断向细胞膜迁移,而在耐药细胞中PSF则未发生膜的迁移,过表达的PSF几乎全部积累在胞内.我们进一步从PSF伴侣蛋白角度探究PSF可能的易位机制,基于此目的,我们构建了一个SBP和PSF全长基因的融合表达载体,过表达的SBP-PSF融合蛋白通过和"链亲和素磁珠"的结合,极大提高了共沉淀PSF天然互作蛋白的效率.经鉴定,细胞角蛋白K18是PSF在髓系白血病细胞内的伴侣蛋白.

细胞角蛋白是一个很大的蛋白家族,在细胞中的表达丰度很高.不同型角蛋白之间通过非共价作用形成杂聚体构成中间丝蛋白,从质膜弥散到细胞浆中维持细胞结构[16].在调节细胞功能方面,角蛋白和多种蛋白形成复合物参与调控细胞周期,凋亡,细胞抗压反应,蛋白合成和跨膜运输等过程[17].受分化和组织类型的调控,不同组织中有不同的角蛋白配对表达.K18 的异常表达参与蛋白质定位,蛋白质靶向/运输和细胞凋亡等过程的调节[18],与多种实体瘤的耐药,侵袭和转移过程高度相关[19,20].研究发现,过表达K18能显著增加细胞膜胆囊纤维化膜转导调节子(cystic fibrosis transmembrane conductance regulator, CFTR) 的表达水平,其通过和CFTR的C末端疏水区互作促进CFTR的膜表达,这个过程不改变CFTR的生物合成,成熟和内化[9],该研究中所展示的K18促进CFTR的膜表达作用,与本研究中K18在敏感株中与PSF互作协助其向细胞膜迁移定位的作用非常相似.我们敲减K18后发现敏感株细胞膜PSF表达率显著下调,进一步证实了敏感株中K18帮助PSF易位细胞膜表达的作用.有趣的是,耐药株中PSF和K18的表达水平皆高于敏感株,但是不发生二者间的相互作用,这是PSF在耐药株中膜易位水平低,在胞内发生积累的主要机制.上述结果提示,在敏感细胞株中,K18通过和PSF发生蛋白质间的相互作用介导PSF向细胞膜转运,缓解PSF在胞内堆积的压力以维持化疗敏感性;而在耐药株中,PSF和K18无相互作用,导致PSF易位障碍.或许正是PSF在细胞内的积累介导了肿瘤的耐药性,这为我们今后开展关于PSF的耐药机制研究提供了方向,在以后的研究中,我们将进一步对PSF在细胞内调控耐药的机制做深入研究.

Funding Statement

国家自然科学基金（81670161）; 北京医院院级科研课题（BJ-2015-105）

Supported by the National Natural Science Foundation of China(81670161); Research Projects of Beijing Hospital(BJ-2015-105)

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